Microorganism-produced enzymes are widely used as a class of biocatalytic reagents in production of synthetic, aromatic, aliphatic and alicyclic aldehydes and alcohols are useful chemical intermediates in chemical, agrochemical, pharmaceutical and food industries. These enzymes are useful in a wide variety of reactions including, e.g., oxidations, reductions, hydrolyses, and carbon—carbon bond ligations.
Biocatalysts are valued for their intrinsic abilities to bind organic substrates and to catalyze highly specific and selective reactions under the mildest of reaction conditions. These selectivities and specificities are realized because of highly rigid interactions occurring between the enzyme active site and the substrate molecule. Biocatalytic reactions are particularly useful when they may be used to overcome difficulties encountered in catalysis achieved by the use of traditional chemical approaches.
Carboxylic acid reductases are complex, multicomponent enzyme systems, requiring the initial activation of carboxylic acids via formation of AMP and often coenzyme A intermediates (see, e.g., Hempel et al., Protein Sci. 2:1890-1900 (1993). Chemical methods for carboxylic acid reductions are generally poor usually requiring prior derivatization and product deblocking with multifunctional reactants.
An enzymatic reaction offers significant advantages over existing methods used in chemical reductions of carboxylic acids, or their derivatives. Unlike many substrates subjected to biocatalytic reactions, carboxylic acids are generally water soluble, rendering them of potentially broad application to this class of enzyme. The carboxylic acid reduction reaction appears to bear the usual desirable features of functional group specificity. It also functions well under mild reaction conditions and produces a high yield of product. The reduction of the activated carboxylic acid intermediate occurs step-wise to give aldehyde products (Gross et al., Eur. J. Biochem. 8:413-419; 420-425 (1969); Gross, Eur. J. Biochem. 31:585-592 (1972)).
The reduction of carboxylic acids by microorganisms is a relatively new biocatalytic reaction that has not yet been widely examined or exploited. Jezo and Zemek reported the reduction of aromatic acids to their corresponding benzaldehyde derivatives by Actinomycetes in Chem. Papers 40(2):279-281 (1986). Kato et al. reported the reduction of benzoate to benzyl alcohol by Nocardia asteroides JCM 3016 (Agric. Biol. Chem. 52(7):1885-1886 (1988)), and Tsuda et al. described the reduction of 2-aryloxyacetic acids (Agric. Biol. Chem. 48(5): 1373-1374 (1984)) and arylpropionates (Chem. Pharm. Bull. 33(11):4657-4661 (1985)) by species of Glomerella and Gloeosporium. Microbial reductions of aromatic carboxylic acids, typically to their corresponding alcohols, have also been observed with whole cell biotransformations by Clostridium thermoaceticum (White et al., Eur. J. Biochem. 184:89-96 (1989)), and by Neurospora (Bachman et al., Arch. Biochem. Biophys. 91:326 (1960)). More recently, carboxylic acid reduction reactions have reportedly been catalyzed by whole cell preparations of Aspergillus niger, Corynespora melonis and Coriolus (Arfmann et al., Z. Naturforsch 48c:52-57 (1993); cf., Raman et al., J. Bacterial 84:1340-1341 (1962)), and by Nocardia asteriodes (Chen and Rosazza,. Appl. Environ. Microbiol. 60(4):1292-1296 (1994)).
Biocatalytic reductions of carboxylic acids are attractive to traditional chemical catalysis because the substrates are water soluble, blocking chemistry is not necessary, reductions are enantioselective (7), and the scope of the reaction is very broad (23, 32).
Aldehyde oxidoreductases are also known as carboxylic acid reductases (CAR), require ATP, Mg2+, and NADPH as cofactors during carboxylic acid reduction (15, 16, 20, 23). The reduction reaction is a stepwise process involving initial binding of both ATP and the carboxylic acid to the enzyme, to form mixed 5′-adenylic acid-carbonyl anhydride intermediates (8, 14, 24, 26, 40) that are subsequently reduced by hydride delivery from NADPH to form the aldehyde product (15, 24).
Aromatic carboxylic acid reductases have been purified to homogeneity only from Neurospora (16) and Nocardia (20, 23). Although details of N— and internal amino acid sequences have been reported for the Nocardia asteriodes enzyme (23), complete gene sequences for these or any other carboxylic acid reductases are unknown.
It is an object of the present invention to provide a purified and isolated bacterial carboxylic acid reductase (CAR) gene and the protein encoded thereby.
It is yet another object of the invention to provide homologous nucleotide sequences and/or amino acid sequences which encode CAR.
It is yet another object of the invention to provide recombinant DNA using expression constructs, vectors, and recombinant cells using the sequences of the invention for production of recombinant CAR.
It is yet another object of the invention to provide for large scale production of and recovery of recombinant CAR, for use in production of synthetic, aromatic, aliphatic and alicyclic aldehydes and alcohols.
It is yet another embodiment of the invention to provide methods of synthesis of chemical compounds such as those for biocatalytically reducing a carboxylic acid, or a derivative thereof, to its corresponding aldehyde product(s), to provide a method of biocatalytically reducing a carboxylic acid, or a derivative thereof, to its corresponding intermediary by-product(s), as exemplified by acyl-AMP analogs, or to provide a method of biocatalytically reducing vanillic acid, or a precursor or derivative thereof, to vanillin, all using recombinant CAR as described the invention disclosed herein.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art on examination of the following, or may be learned by practice of the invention.